Spinal muscular atrophy (SMA) is characterized by degeneration of the anterior horn cells of the spinal cord, leading to progressive symmetrical limb and trunk paralysis and muscular atrophy. SMA is the second most common fatal autosomal recessive disorder, second only to cystic fibrosis, and the most common genetic cause of childhood mortality affecting 1 in 6,000 newborns (Roberts et al., 1970, Arch. Dis. Child. 45:33-38; Pearn, 1973, J. Med. Genet. 10:260-265; Pearn, 1978, J. Med. Genet. 15:409-413; Czeizel and Hamular, 1989, J. Med. Genet. 21:761-763). Childhood spinal muscular atrophies are divided into severe (type I, Werdnig-Hoffman disease) and mild forms (type II and III) according to the age of onset and the severity of the disease (Munsat, 1991, Neuromusc. Disord. 1:81; Crawford and Pardo, 1996, Neurobiol. Dis. 3:97-110). The Survival of Motor Neurons (SMN) gene (Lefebvre et al., 1995, Cell 89:155-165) has been shown to be the SMA disease gene, and it is deleted or mutated in over 98% of SMA patients (Bussaglia et al., 1995, Nat. Genet. 11:335-337; Chang et al., 1995, Am. J. Hum. Genet. 57:1503-1505; Cobben et al., 1995, Am. J. Hum. Genet. 57:805-808; Hahnen et al., 1995, Hum. Mol. Genet. 4:1927-1933; Hahnen et al., 1996, Am. J. Hum. Genet. 59:1057-1065; Lefebvre et al., 1995, Cell 89:155-165; Rodrigues et al., 1995, Hum. Mol. Genet. 4:631-634; Velasco et al., 1996, Hum. Mol. Genet. 5:257-263; Lefebvre et al., 1997, Nat. Genet. 16:265-269).
Two inverted gene copies of the SMN gene are located in a 500 kb inverted repeat at chromosome 5q13. In over 98% of all SMA patients, the telomeric copy of SMN (SMNT) is deleted or mutated while the centromeric copy of the gene (SMNC) is unaffected (Lefebvre et al., 1995, Cell 89:155-165). The SMN gene encodes a protein of about 296 amino acids having a molecular mass of approximately 40 kDa. The sequence of the protein does not exhibit any significant homology to any other protein of known function in the currently available protein databases.
Recently, in the course of studies of the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs) (Dreyfuss et al., 1993, Ann. Rev. Biochem. 62:289-321), it was found that the SMN protein interacts with fibrillarin, an RNA-binding protein involved in rRNA processing, and with several other RNA-binding proteins (Liu and Dreyfuss, 1996, EMBO J. 15:3555-3565). Monoclonal antibodies to SMN localized the protein to a unique cellular location. SMN exhibits a general localization in the cytoplasm and is particularly concentrated in several prominent nuclear bodies called gems (for gemini of coiled bodies). Gems are novel nuclear structures which are related in number and size to coiled bodies and are usually found in close proximity to them (Liu and Dreyfuss, 1996, EMBO J. 15:3555-3565). Coiled bodies, which were first described by Ramón y Cajal (1903, Trab. Lab. Invest. Biol. 2:129-221), are prominent nuclear bodies found in widely divergent organisms, including plant and animal cells (Bohmann et al., 1995, J. Cell Sci. 19:107-113; Gall et al., 1995, Dev. Genet. 16:25-35). Coiled bodies contain the spliceosomal U1, U2, U4/U6, and U5 snRNPs, U3 snoRNAs, and several proteins, including the specific marker p80-coilin, fibrillarin, and NOP140 (Bohmann et al., 1995, J. Cell Sci. 19:107-113, and references therein; Gall et al., 1995, Dev. Genet. 16:25-35). Expression of p80-coilin mutants and microscopic observations suggests a close association between coiled bodies and the nucleolus (Raska et al., 1990, J. Struct. Biol. 104:120-127; Andrade et al., 1991, J. Exp. Med. 173:1407-1419; Bohmann et al., 1995, J. Cell Biol. 131:817-831). However, the specific functions of coiled bodies are not clear. Current ideas propose that coiled bodies may be involved in processing, sorting, and assembly of snRNAs and snoRNAs in the nucleus. The close association of gems and coiled bodies raises the possibility that the SMN protein and gems are also involved in the processing and metabolism of small nuclear RNAs (Liu and Dreyfuss, 1996, EMBO J. 15:3555-3565).
The Sm class of small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6, and U5 are major constituents of the spliceosome, the catalytic center of the pre-mRNA splicing reaction (Moore et al., 1993, In: The RNA World, pp. 303-358, Gesteland and Atkins, eds., Cold Spring Harbor Laboratory Press, Plainview, NY; Madhani and Guthrie, 1994, Annu. Rev. Genet. 28:1-26). Each spliceosomal snRNP consists of one (U1, U2, and U5) or two (U4/6) snRNAs, a common set of at least eight Sm proteins, termed B, B′, D1, D2, D3, E, F, and G, and specific polypeptides that are associated with only one individual U snRNP (reviewed by Lührmann et al., 1990, Biochim. Biophys. Acta Gene Struct. Express. 1087:265-292). With the exception of U6, all spliceosomal snRNAs share two structural features: the 5′-terminal trimethylguanosine (m3G) cap and a short, single-stranded, eight-to-ten nucleotide uridine-rich sequence flanked by two hairpin loops, referred to as the Sm site (Branlant et al., 1982, EMBO J. 1:1259-1265; Reddy and Busch, 1988, In: Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles, pp. 1-37, Birnstiel, ed., Springer-Verlag, Berlin). The Sm site is the primary binding site for the Sm proteins. The remaining snRNA domains provide binding sites for the snRNA-specific snRNP proteins and for RNA-RNA interactions (Lührmann et al., 1990, Biochim. Biophys. Acta Gene Struct. Express. 1087:265-292). U6 differs from the other spliceosomal U snRNAs in that it contains a γ-monomethyl cap instead of the (m3G) cap and does not bind directly to Sm proteins due to its lack of an Sm site (Reddy and Busch, 1988, supra; Singh and Reddy, 1989, Proc. Natl. Acad. Sci. USA 86:8280-8283). The snRNP-specific proteins have snRNP-specific functions in the splicing reaction. In contrast, the only known function for the Sm proteins is in the biogenesis of U snRNPs.
The biogenesis of snRNPs, which is illustrated in FIG. 26 herein, is a complex, multistep process (DeRobertis, 1983, Cell 32:1021-1025; Fisher et al., 1985, Cell 42:751-758; Mattaj, 1988, In: Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles, pp. 100-114, Birnstiel, ed., Springer-Verlag, Berlin; Feeney et al., 1989, J. Biol. Chem. 264:5776-5783; Neuman de Vegvar and Dahlberg, 1990, Mol. Cell. Biol. 10:3365-3375; Zieve and Sauterer, 1990, Crit. Rev. Biochem. Mol. Biol. 25:1-46). Spliceosomal snRNAs that contain the Sm site are first exported to the cytoplasm, where they associate with the Sm proteins (B, B′, D1, D2, D3, E, F, and G) (Mattaj and DeRobertis, 1985, Cell 40:111-118). Next, in a reaction that requires the assembled Sm core domain (comprising the Sm proteins bound to the Sm site), the 7-methylguanosine (m7G) cap of the snRNAs is hypermethylated to yield 2,2,7-trimethylguanosine (m3G) (Mattaj, 1986, Cell 46:905-911). In addition, varying numbers of nucleotides are trimmed from the 3′ end of several of the snRNAs. Proper Sm core assembly, cap hypermethylation, and 3′-end processing are important for nuclear import of the assembled snRNP particles (Fischer and Lührmann, 1990, Science 249:786-790; Hamm et al., 1990, Cell 62:569-577). Finally, just before or after the nuclear import, many internal nucleotides are modified and more than 30 snRNP-specific proteins associate with the individual snRNP precursors to complete their biogenesis (Mattaj, 1988, In: Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles, pp. 100-114, Bimstiel, ed., Springer-Verlag, Berlin; Lührmann et al., 1990, Biochim. Biophys. Acta Gene Struct. Express. 1087:265-292; Neuman de Vegvar and Dahlberg, 1990, Mol. Cell. Biol. 10:3365-3375; Zieve and Sauterer, 1990, Crit. Rev. Biochem. Mol. Biol. 25:1-46). However, the detailed mechanism of how the Sm core proteins and the snRNP-specific proteins form functional assembled snRNPs is not clear. There is, to date, no effective treatment for SMA and the mechanism underlying the disease process is poorly understood. Thus, there is an acute and long-felt need to understand the mechanism of the disease process and, more importantly, for the development of methods of treating this common and usually fatal disease. The present invention addresses these needs.